1. Field of the Invention
The present invention relates to glutaminase and a glutaminase gene encoding the same, more specifically, to a protein having a glutaminase activity and excellent in salt resistance and thermostability, and a gene encoding the protein having a glutaminase activity.
2. Prior Art
Glutaminase is an enzyme which generates ammonia and L-glutamic acid which gives good taste by decomposing L-glutamine. Glutaminase has an important role in a food industry and is useful for producing, for example, soy sauce or a cooked product obtained by enzymatically decomposing protein. Glutaminase has been isolated from various kinds of biological species and its enzymological properties and the gene have been reported (e.g., Japanese Patent Publication No. 38748/1994).
In the preparation of soy sauce and the preparation of a cooked food containing a large amount of salt, glutaminase excellent in an optimum pH, salt resistance and thermostability has been desired. A group to which the present inventors have belonged has previously found a novel glutaminase which is excellent in salt resistance and thermostability, and can effectively produce a protein-hydrolyzed product (e.g., soy sauce) enriched in an amount of glutamic acid from Cryptococcus nodaensis G60 (FERM BP-6351 deposited on May 13, 1998 with the National Institute of Advanced Industrial Science and Technology, Japan) (Japanese Provisional Patent Publication No. 332553/1999 which corresponds to U.S. Pat. No. 6,063,409 herein incorporated by reference).
For further improving the property of the above enzyme by a genetic engineering means and for producing the enzyme with a large amount, it is important to obtain a gene of the enzyme.
According to the above, it is possible to improve qualities of the protein-hydrolyzed product (e.g., soy sauce) easily and provide the same with inexpensive.
An object of the present invention is to provide a protein having a glutaminase activity and excellent in salt resistance and thermostability, and a gene encoding the same.
The present inventors have earnestly investigated about the above-mentioned problems in various manners and as a result, they have succeeded in isolating a glutaminase gene derived from Cryptococcus nodaensis to accomplish the present invention.
That is, the present invention provides the following materials and process.
1. A protein shown in either of the following (a) or (b):
(a) a protein having an amino acid sequence represented by amino acid numbers 1 to 684 shown in SEQ ID NO:2,
(b) a protein having a glutaminase activity in which one or more amino acids is/are deleted from, substituted by, inserted to or added to the amino acid sequence of the above-mentioned (a).
2. A protein shown in either of the following (c) or (d):
(c) a protein having an amino acid sequence represented by amino acid numbers 49 to 684 shown in SEQ ID NO:2,
(d) a protein having a glutaminase activity in which one or more amino acids is/are deleted from, substituted by, inserted into or added to the amino acid sequence of the above-mentioned (c).
3. A gene containing DNA shown in either of the following (e) or (f):
(e) a gene containing DNA encoding the protein according to claim 1,
(f) a gene encoding a protein which hybridizes with the DNA of the above-mentioned (e) under a stringent condition and has a glutaminase activity.
4. A gene containing DNA shown in either of the following (g) or (h):
(g) a gene containing DNA encoding the protein according to claim 2,
(h) a gene encoding a protein which hybridizes with the DNA of the above-mentioned (g) under a stringent condition and has a glutaminase activity.
5. A recombinant DNA containing the gene described in the above 3 or 4.
6. A transformant or a transductant containing the recombinant DNA described in the above 5.
7. A process for producing glutaminase which comprises culturing the transformant or the transductant described in the above 6 and collecting glutaminase from a culture medium.
In the following, the present invention will be explained in detail.
1. A Protein Having a Glutaminase Activity and a Gene Encoding the Same
The protein of the present invention is a protein shown in either of the following (a) or (b).
(a) a protein having an amino acid sequence represented by amino acid numbers 1 to 684 shown in SEQ ID NO:2,
(b) a protein having a glutaminase activity in which one or more amino acids is/are deleted from, substituted by, inserted to or added to the amino acid sequence of the above-mentioned (a).
The protein shown in (a) can be obtained by subjecting a natural type glutaminase gene derived from a chromosomal DNA or cDNA of Cryptococcus nodaensis G60 to cloning and introducing the resulting clone into a suitable host-vector system to express the same.
Incidentally, one or a plural number of the amino acids may be deleted from, substituted by, inserted into or added to the amino acid sequence of the above (a) so long as it has a glutaminase activity as shown in the above (b). In the present specification, xe2x80x9ca plural numberxe2x80x9d generally means 2 to 300, preferably 2 to 170, more preferably 2 to 50, most preferably 2 to 10 amino acids whereas it is different depending on a position in a steric structure or a kind of the amino acid residue.
Such a mutant type glutaminase, i.e., the protein of the above (b) can be obtained by introducing variation such as substitution, deletion, insertion, addition or inversion into the base sequence of the natural type glutaminase gene to prepare a variant type glutaminase gene, and introducing the gene into a suitable host-vector system to express the same.
As a method of introducing variation into a gene, there may be mentioned, for example, a site-specific mutation introducing method, a random mutation introducing method by PCR, and a method in which a gene is selectively cleaved and then a selected nucleotide is removed or added, and the cleaved genes are linked.
The glutaminase gene of the present invention is a gene containing DNA encoding the protein of the above (a) or (b). Incidentally, the glutaminase gene of the present invention may be a gene encoding a protein having a glutaminase activity which hybridizes with the DNA encoding the protein of the above-mentioned (a) or (b) under a stringent condition. In the present specification, xe2x80x9ca stringent conditionxe2x80x9d means, for example, a condition wherein a sodium concentration is 50 to 300 mM, preferably about 150 mM and a temperature is 42 to 68xc2x0 C., preferably about 65xc2x0 C.
Examples of the gene containing DNA encoding the protein of the above-mentioned (a) may include DNA containing base sequence represented by the base numbers 1 to 2052 shown in SEQ ID NO:1 in the sequence listing. This DNA is a natural type glutaminase gene.
The natural type glutaminase gene can be obtained by subjecting a natural type gene derived from a chromosomal DNA or CDNA of Cryptococcus nodaensis G60 to cloning. As a method of go subjecting to cloning of the gene, for example, there may be mentioned a method in which glutaminase is purified and a partial amino acid sequence is determined, then, a suitable probe DNA is synthesized and screening is carried out from the chromosomal DNA of Cryptococcus nodaensis by using the probe DNA. Also, there may be mentioned a method in which a suitable primer DNA is prepared based on a partial amino acid sequence, and the DNA containing a fragment of the gene is amplified by a polymerase chain reaction (hereinafter abbreviated to as xe2x80x9cPCR methodxe2x80x9d) such as the 5xe2x80x2 RACE method and the 3xe2x80x2 RACE method, and the resulting genes are linked to obtain DNA containing whole length gene.
In more detail, a natural type glutaminase gene can be obtained as mentioned below. First, Cryptococcus nodaensis is cultured, and after the resulting culture broth is lyophilized in a liquid nitrogen, it is physically ground by using a mortar, etc., to obtain fine powder state cell pieces, and chromosomal DNA is extracted from the cell pieces by the conventional manner. In the extraction operation, a commercially available DNA kit can be utilized.
Then, glutaminase is purified to determine the N-terminal amino acid sequence. Moreover, an amino acid sequence of a peptide fragment obtained by digestion using lysylendopeptidase is determined.
Glutaminase can be purified by the method as disclosed in Japanese Provisional Patent Publication No. 332553/1999. That is, Cryptococcus nodaensis G60 is firstly inoculated into a suitable medium to obtain a culture broth containing proliferated cells. After adding a cell wall lytic enzyme to the cells obtained by centrifugation of the culture broth, the mixture was centrifuged to obtain a supernatant. The supernatant is heated to denature impurity proteins and the resulting material is further centrifuged to remove denatured proteins.
To the above-mentioned supernatant is added acetone (xe2x88x9220xc2x0 C.), the mixture is well stirred and after maintaining the mixture at 4xc2x0 C. for 5 hours, the mixture is centrifuged to collect precipitate. The precipitate is dissolved in an acetate buffer to obtain a crude enzyme solution containing glutaminase. Moreover, a fraction having glutaminase activity is purified by using a DEAE-Sepharose column, a phenyl-Sepharose column, a hydroxyapatite column, a gel filtration column, an HPLC (high performance liquid chromatography), etc., whereby glutaminase can be purified.
Then, a primer to be used for PCR is synthesized in view of an information about a partial amino acid sequence, a codon use frequency of microorganisms belonging to Cryptococcus genus, and the like. Next, PCR is carried out by using these primers and the chromosomal DNA obtained as mentioned above as templates. Moreover, based on the base sequence of the resulting DNA, a primer is synthesized.
Next, the chromosomal DNA is treated by a restriction enzyme present at a part of a glutaminase gene region (an intron is included therein) obtained, and then, self ligation is carried out. Using the resulting material as a template, inverse PCR is carried out by using the above-mentioned primer. The base sequences of the respective DNA fragments thus obtained are ligated to obtain a glutaminase gene containing an intron which is encoded on a genome.
Next, a glutaminase gene containing no intron, i.e., cDNA encoding the glutaminase gene is obtained. First, Cryptococcus nodaensis G60 is cultured, and after lyophilizing the resulting cells in liquid nitrogen, the cells are ground by using a mortar, etc., to obtain fine powder state cell pieces. Then, whole RNA fractions are extracted from the cell pieces by the conventional manner. In the extraction operation, a commercially available RNA kit can be utilized.
RNA is recovered from the resulting RNA extracted solution by ethanol precipitation, and RNA having a poly-A chain may be fractionated from the recovered RNA by the conventional manner. In the fractionation operation, a commercially available Oligo dT column can be utilized.
Next, primers to be used in PCR are synthesized from an N-terminal amino acid sequence of glutaminase and an amino acid sequence of a peptide fragment obtained by digestion using lysylendopeptidase. By using this primer DNA and the RNA obtained as mentioned above, DNA""s containing fragments of the gene are amplified by a suitable RT-PCR reaction such as the 5xe2x80x2 RACE method and the 3xe2x80x2 RACE method, and ligated these to obtain DNA containing whole gene. In the partial cDNA synthesis operation by the 5xe2x80x2 RACE method and the 3xe2x80x2 RACE method, a commercially available kit can be utilized.
DNA is amplified by using the above-mentioned cDNA as a template and subjecting to PCR using synthetic primers complementary to the 5xe2x80x2-terminus sequence and the 3xe2x80x2-terminus sequence. The amplified DNA can be subjected to cloning according to the conventional manner.
A recombinant DNA can be obtained by inserting the amplified DNA into a suitable vector. In the cloning, a commercially available kit such as TA Cloning Kit (trade name, available from Invitrogen Co.), a commercially available plasmid vector DNA such as pUC119 (trade name, available from Takara Shuzo Co.), pBR322 (trade name, available from Takara Shuzo Co.), pBluescript SK+ (trade name, available from Stratagene Co.), a pCR2.1-TOPO vector using TOPO TA Cloning Kit (trade name, manufactured by Invitrogen Co.), etc., and a commercially available bacteriophage vector DNA such as xcexEMBL3 (trade name, available from Stratagene Co.), etc. can be used.
By using the resulting recombinant DNA, a transformant or a transductant can be obtained by transforming or transducing a host cell therein, respectively. Transformation can be carried out, for example, by the method of D. M. Morrison (Method in Enzymology, 68, 326-331, 1979). Also, transduction can be carried out, for example, by the method of B. Hohn (Method in Enzymology, 68, 299-309, 1979).
As a host cell, microorganisms such as Escherichia coli (K-12, Escherichia coli JM109 (trade name, available from Takara Shuzo Co.), XL-Blue (trade name, available from Funakoshi Co.)), yeast (INVSc1 (trade name, available from Invitrogen Co.), filamentous fungi, actinomycetes, etc., and animal cells can be used.
The whole or total base sequence (see SEQ ID NO:1) of the DNA amplified as mentioned above can be analyzed by using, for example, LI-COR MODEL 4200L sequencer (trade name, manufactured by LI-COR, Inc.), 370 DNA sequence system (trade name, manufactured by Perkin-Elmer Co.), and the like. By comparing the base sequence with an information of a partial amino acid sequence, it can be confirmed whether a natural type glutaminase gene can be obtained or not.
According to the analyses of the natural type glutaminase gene, translated polypeptide, i.e., an amino acid sequence of the protein as mentioned (a) can be fixed.
In another embodiment, the glutaminase of the present invention is a protein shown in either of the following (c) or (d).
(c) a protein having an amino acid sequence represented by amino acid numbers 49 to 684 shown in SEQ ID NO:2,
(d) a protein having a glutaminase activity in which one or more amino acids is/are deleted from, substituted by, inserted into or added to the amino acid sequence of the above-mentioned (c) .
The protein shown in (c) is a protein found by the present inventors to present in a glutaminase purified standard product of Cryptococcus nodaensis. This protein can be obtained by introducing variation of deletion into the base sequence of the natural type glutaminase gene to prepare a variant type glutaminase gene which encodes an amino acid sequence shown by the amino acid numbers 49 to 684 of SEQ ID NO:2, and introducing the same into a suitable host-vector system to express the same.
The variant type glutaminase gene encoding the protein shown in (c) can be specifically prepared by the method as mentioned below.
A sense primer is synthesized in a form of linking abase sequence encoding a protein with amino acid numbers 49 to 684 of SEQ ID NO:2 to the downstream of the known signal sequence. Then, an antisense primer which complements to the base encoding the C-terminal amino acid sequence is synthesized. To the 5xe2x80x2-terminuses of the respective primers, suitable restriction enzyme recognition sites are added in a form that their frames are adapted thereto. The amplified fragments are incorporated into the cloning vector as described above. According to the above procedure, a variant type glutaminase gene encoding the protein shown in (c).
Incidentally, in the protein, as shown in (d), one or a plural number of amino acids may be deleted, substituted, inserted or added in the amino acid sequence of (c) so long as it has a glutaminase activity. The protein shown in (d) can be obtained by introducing variation of deletion into the base sequence of DNA encoding the natural type glutaminase gene or the protein shown in (c) to prepare a variant type glutaminase gene, and introducing the gene into a suitable host-vector system to express the same.
As an example of the gene containing DNA encoding the protein shown in the above (c), there may be mentioned DNA containing a base sequence shown by the base numbers 145 to 2052 of SEQ ID NO:1 in the Sequence Listing. Incidentally, the glutaminase gene of the present invention may be a gene which hybridizes with the DNA encoding the protein having a glutaminase activity of the above-mentioned (c) or (d) under a stringent condition.
2. Preparation Method of Glutaminase
When the glutaminase of the present invention is to be prepared, a recombinant DNA containing the glutaminase gene is firstly prepared. Then, a transformant or a transductant containing the recombinant DNA is prepared and cultured, and glutaminase is collected from a culture medium.
To produce the protein having a glutaminase activity by using the glutaminase gene of the present invention, it is necessary to select a suitable host-vector system. Such a system may include a system of yeast expression vector pYES2 (trade name, available from Invitrogen Co.) and yeast (Saccharomyces cerevisiae), a system of Escherichia coli expression vector pTE (trade name, available from Stratagene Co.) and Escherichia coil (E. coli) and the like. The system of yeast is preferably used in the point that saccharide chain addition to the protein occurs.
The recombinant DNA can be obtained by inserting the glutaminase gene into a suitable vector. As the vector, there may be used, for example, yeast expression vector pYES2, pYD1 (both trade names, available from Invitrogen Co.), pAUR123 (trade name, available from Takara Shuzo, Co.), pYEX-BX, pYEX-S1, PYEX-4T (all trade names, available from CLONETECH Co.), YEpFLAG-1 (trade name, available from SIGMA Co.), Escherichia coli expression vector pSET (trade name, available from Invitrogen Co.), pTE (trade name, available from Stratagene Co.), and the like.
Then, the recombinant DNA is transformed or transduced in a host cell. Transformation into yeast can be carried out, for example, by the method of D. M. Becker et al. (Method in Enzymology, 194, 182-187, 1991). Transformation into Escherichia coli can be carried out, for example, by the method of B. Hohn (Method in Enzymology, 68, 299-309, 1979). As a host cell, microorganisms such as Escherichia coli, yeast, filamentous fungi, actinomycetes, etc., and animal cells can be used.
According to the above procedure, a transformant or a transductant having an ability of producing glutaminase can be obtained. To culture the transformant or the tranductant, they may be cultured by the usual solid culture method, and a liquid culture method is preferably used if the situation allows.
When yeast is used as a host cell, as a medium, a generally employed nutrient-rich medium such as YPD medium and YM medium can be used. Also, when a selective medium is used depending on the genetic properties of the host cell, a SD medium which is the minimum medium can be used. When the selective medium is used, a selective pressure varies depending on the selected vector-host system to be used so that an amino acid(s), a nucleic acid(s) and the like other than the selective pressure is/are added to the minimum medium depending on the genetic requirements of the host cell.
In addition, an inorganic salt(s), a starting material of saccharide(s), vitamin(s) and the like may be optionally added to the medium, depending on necessity. Incidentally, an initial pH of the medium is preferably adjusted to pH 6 to 9. Moreover, some of the vectors can control expression of a protein. When these vectors are used, an inducer corresponding to the vector, such as galactose, a copper ion, etc., is added to the medium whereby glutaminase can be induced.
When yeast is cultured, culture is carried out at 25 to 35xc2x0 C., preferably about 30xc2x0 C. for 24 to 48 hours by an aeration stirring deep culture, shaking culture, standing culture, and the like.
Incidentally, the genetic engineering method in the present invention can be carried out, for example, according to the descriptions such as xe2x80x9cMolecular Cloning: A Laboratory Manual 2nd Editionxe2x80x9d (1989), Cold Spring Harbor Laboratory Press, ISBN 0-87969-309-6, xe2x80x9cCurrent Protocols in Molecular Biologyxe2x80x9d (1989), John Wiley and Sons, Inc. ISBN 0-471-50338-X, etc.